Mechanisms for the in vivo migration of cells across native tissue barriers have been studied through various in vitro assay methods devised to provide interaction between living cells and diverse tissue cultures. An important focal point of such research has been the evaluation of mechanisms by which metastatic cancer cells arising in glandular or squamous epithelium traverse native tissue barriers such as collagen/stroma barriers to enter the circulatory and lymphatic systems. Other studies in this area have been directed to migration of anti-inflammatory cells such as leukocytes from the blood vessels to the site of injury. Broadly, both the morphological and biochemical aspects of cellular migration are sought to be understood by appropriate bioassay techniques, with particular reference to histological migration routes, including mechanisms of tissue penetration and repair, and the role of chemoattractants in inducing migration through native tissue barriers. Another related area of investigation comprises the interaction of nonmigrating normal or malignant cells with these tissue barriers.
Known prior art bioassay methods for cellular interaction with native tissue barriers have generally proved to be unsatisfactory from a qualitative or quantitative standpoint, or both, primarily due to the tissues employed or their preparation. In conventional methods for in vitro assessment of cellular migration of tissues, whole tissue fragments of, for example, chick embryonic skin or mouse lung have been employed. Typically, these fragments are merely randomly cultured with the cellular material, making a reliable qualitative analysis of results difficult, and a quantitative analysis of results frequently impossible. For example, chick embryonic skin is commonly employed as the tissue medium in cellular migration assay techniques. This skin is a relatively thick complex of varying tissues, and includes native cellular components such as host-immune and inflammatory cells. Many of these tissue and cellular components are extraneous to in vivo cellular migration in humans, and serve only to impede or actively interfere with the mechanisms of interest. Chick embryonic skin is conventionally used in assays as tissue fragments intermixed with cells and a reliable evaluation of results is usually not possible without complete examination of each fragment, since sites of interaction of cells and tissue are not uniformly disposed over the tissue. Further, a quantitative analysis of tissue penetration cannot be made with chick embryonic skin and similar conventional tissues due to the thickness of the substrate and the presence of anomalous tissue. Other tissues commonly employed in similar assay techniques, including chick blastoderm, chick embryonic heart, chick embryonic mesonephros, chick wing bud, and extracted bovine articular cartilage, have similar disadvantages, including the major disadvantage of being of non-human origin. Known uses of human tissue, specifically human decidual tissue, have been relatively unsuccessful. While quantitative assay methods employing non-fragmented tissue have been developed, such known prior art methods have employed complex tissues of non-human origin. For example, radioactively labelled tumor cells have been cultured within the lunea of a perfused canine vein or cultured on chicken chorioallantoic membrane; the proportion of tumor cells which traverse these tissue barriers is calculated by measuring the radioactivity on either side of the barrier. Neither of these assay systems lends itself to the routine performance of assays on a useful scale; further, the chorioallantoic membrane contains a plurality of multicellular and connective tissue layers, and also is vascularized, so that host-immune cells are present.
It is accordingly an object of the present invention to provide an in vitro assay method for cellular interaction with natural tissue barriers wherein the tissue employed presents a normal connective tissue matrix barrier including attached intact cell layers.
It is another object of this invention to provide tissue for use in in vitro assay techniques for cellular interaction with normal tissue barriers which permits a reliable qualitative and quantitative analysis of the morphological and biochemical aspects of cellular interaction with native tissue barriers, including mechanisms of cellular penetration and repair and influences of chemotactic and chemokinetic factors.
It is a further object of the invention to provide a natural barrier tissue free from extraneous tissue and cellular components for use in in vitro assays which is readily available in large, uniform sheets as living tissue of human origin.
It is yet another object of this invention to provide a natural barrier tissue for in vitro assays of in vivo cellular migration which substantially duplicates native human tissue barriers.
It is an additional object of this invention to provide an in vitro qualitative and quantitative assay method for the identification and characterization of invasive cells, particularly malignant tumor cells, and also to provide diagnostic apparatus for use in the identification of metastatic tumor cells.
It is yet another object of this invention to provide a tissue of human origin readily available in the form of a large intact membrane for use as a substrate in the culture of normal cells.
Further objects and advantages of the invention will be apparent from the disclosure herein.